Fig. 6.

GATA4 is necessary for ERα recruitment to estrogen-responsive enhancers in osteoblasts. A, U2OS-ERα cells were transfected with either an siRNA directed at luciferase (siLUC) or GATA4 (siGATA4). At 48 h after transfection, cells were treated for 45 min with 10 nm E2. ChIP was performed with antibodies to ERα, and qPCR was performed to detect the ERα binding sites at the indicated enhancer regions. Each PCR was normalized to input and represented as enrichment over a negative genomic locus [hemoglobin (HBB) promoter]. B, U2OS-ERα cells were deprived of estrogen for 3 d in phenol red-free media containing 5% CDT-FBS. Cells were then treated for 45 min with 10 nm E2. ChIP was performed with antibodies to GATA4, and qPCR was performed to detect the enhancers near the indicated genes. Each PCR was normalized to input and represented as enrichment over a negative genomic locus [hemoglobin (HBB) promoter]. EtOH, Ethanol. C, Cells were treated as in B, and ChIP was performed with and antibody to H3K4me2. Quantitative PCR was performed to detect the indicated enhancer regions. Each PCR was normalized to input and represented as enrichment over a negative genomic locus [hemoglobin (HBB) promoter]. Error bars represent mean ± 1 sd. **, P value < 0.01; *, P value < 0.05 using an unpaired t test.